Paratuberculosis is caused by Mycobacterium avium subsp. paratuberculosis that is one of acid-fast bacteria, and is a chronic granulomatous diarrheal infectious disease in ruminants such as cattle, goats, sheep, and buffalos. With regard to pandemic of bovine paratuberculosis in Japan, the increase of infected animals and the expansion of the outbreak areas have been seen since 1980. In particular, the outbreak numbers increased at a rate of 100 to 200 cattle/year from the 1990s, and exceeded 800 in 2000.
FIG. 1 shows the course of infection (from infection to onset of symptoms) of paratuberculosis and shift in immune response. Paratuberculosis is developed by oral infection of Mycobacterium avium subsp. paratuberculosis in the early period after birth. However, most of the routes of infection are still unclear, and differences in the course of the disease between individuals are larger than those in any other diseases.
The sub-clinical infection stage between infection and onset of symptoms, (clinical signs such as diarrhea are observed), is generally 2 to 5 years or more. In some individuals, the period is about ten years or more, or the symptoms are not found throughout their life. From this standpoint, paratuberculosis is considered as an “ultrachronic infection”.
The ELISA method is a diagnostic method involving detecting a specific antibody against Mycobacterium avium subsp. paratuberculosis, and is prevalent all over the world because of its simplicity (see Momotani Eiichi “Up-to-date information on diagnosis of bovine Johne's disease”, Journal of Clinical Veterinary Medicine, vol. 16 (9), 1998, 24-31, in Japanese). However, the method can be used in diagnosing cattle in advanced stage, or after increase of antibody level, but cannot diagnose sub-clinically infected animals before the specific antibody level increases. However, as a result of prevalence of this ELISA method as a standard for diagnosis of paratuberculosis, sub-clinically infected animals that cannot be diagnosed by the ELISA method relatively increases, although ELISA-positive cattle decreases. Therefore, the diagnosis becomes increasingly harder.
Meanwhile, as shown in FIG. 1, the cell-mediated immunity of an animal infected with Mycobacterium avium subsp. paratuberculosis is induced at the early stage of infection but is then gradually decreased. Examples of diagnostic methods of such cell-mediated immunity include a johnin reaction and an interferon γ (IFNγ) ELISA method.
The Johnin reaction (johnin intradermal reaction) is a diagnostic method of detecting the cell-mediated immunological response of a host against Mycobacterium avium subsp. paratuberculosis and is an intradermal reaction similar to the tuberculin reaction in tuberculosis. That is, the diagnosis is performed by: intradermally injecting a culture supernatant of Mycobacterium avium subsp. Paratuberculosis (Mycobacterium avium subsp. paratuberculosis PPD, Johnin PPD) to the root of the tail of a subject animal; and observing and measuring the redness and swelling at the injection site again. The method has been conventionally used, and also in Japan, this method is employed as a diagnostic method for paratuberculosis in the Domestic Animal Infectious Disease Control Law.
However, johnin reaction requires two visits to a farm for diagnosing and moreover, in the case of many subject animals to be tested, requires more labor and time, so that it is unfavorable for the test of a large number of specimens. Therefore, to detect a specific antibody, the ELISA method tends to be more widely performed in the world than johnin reaction.
On the other hand, the IFNγ ELISA method is a method of detecting the amount of produced IFNγ in vitro utilizing a cell-mediated immunological response against Mycobacterium avium subsp. paratuberculosis (see Billman-Jacobe H, Carrigan M, Cockram F, Corner L A, Gill I J, Hill J F, Jessep T, Milner A R, Wood P R, 1992. A comparison of the interferon gamma assay with the absorbed ELISA for the diagnosis of Johne's disease in cattle. Aust Vet J. 69:25-28). When the peripheral blood of a sub-clinically infected cattle that has been infected with Mycobacterium avium subsp. Paratuberculosis and a cattle that develops the disease are stimulated by a Mycobacterium avium subsp. paratuberculosis antigen, IFNγ that is one of cytokines is produced in large amounts by the sub-clinically infected cattle. Therefore, it was revealed that the amount of produced IFNγ is effective in diagnosis of a sub-clinically infected cattle, (see Stabel J R, 1996. Production of gamma-interferon by peripheral blood mononuclear cells: an important diagnostic tool for detection of subclinical paratuberculosis. J Vet Diagn Invest. 8:345-350) and the present method was introduced in the 1990s.
The IFNγ ELISA method enables more sensitive diagnosis of infection of paratuberculosis than johnin reaction also utilizing a cell-mediated immunological response, but the sensitivity is lowered as the cell-mediated immunity is gradually decreased, resulting in undetectable levels. Meanwhile, various factors (such as the stage of infection and proliferation level of Mycobacterium avium subsp. paratuberculosis in a lesion) may also decrease the sensitivity, so that the method still has problems as a diagnostic method.
Meanwhile, there has also been studied a diagnostic method using, as an index, a phenomenon in which a T-lymphocyte that recognizes a Mycobacterium avium subsp. Paratuberculosis antigen reacts and induces cell proliferation thereof when the lymphocyte is exposed to the antigen again (lymphocyte blastogenesis reaction, lymphocyte proliferation reaction) (see Kreeger J M, Snider T G 3rd., 1992. Measurement of lymphoblast proliferative capacity of stimulated blood mononuclear cells from cattle with chronic paratuberculosis. Am J Vet Res. 53:392-395). However, the method requires use of a radioactive isotope for proliferation of the lymphocyte, so it is not suitable for field diagnostic applications and therefore is not practical.
As described above, the infection period of Mycobacterium avium subsp. paratuberculosis includes a long sub-clinical stage of infection as described above, which further includes an immunological un-responsive period difficult to diagnose the disease when diagnosis cannot be achieved by either of cell-mediated immunity or detection of a specific antibody. Moreover, the period varies greatly from individual to individual and is long (3 to 5 years), so that it is impossible to find infected animals effectively by domestic epidemic prevention or by import quarantine, which makes it difficult to eradicate paratuberculosis. 
An infected animal in the sub-clinical stage of infection has a certain lesion sustainably, and microorganisms are irregularly excreted in feces, so that the feces serve as contamination sources and cause spread of infection. In particular, from the aspect of epidemic prevention, they cause big problems in the immunological un-responsive period difficult to diagnose the disease.
Examples of a diagnostic method to detect such irregular excretion of the bacteria includes isolating and identifying Mycobacterium avium subsp. Paratuberculosis excreted in feces.
However, the method requires several months to culture Mycobacterium avium subsp. paratuberculosis in order to recognize the bacterial colonies. Therefore, it is difficult to diagnose the infection at an early stage, and there is a problem in that the infection is spread owing to excretion of the bacteria from a carrier animal during culture. Meanwhile, excretion of the bacteria occurs irregularly and nonpersistently, so there is also a problem in that some infected individuals are not accurately diagnosed.
It takes a long time to culture Mycobacterium avium subsp. paratuberculosis because Mycobacterium avium subsp. paratuberculosis belongs to the group III atypical acid-fast bacteria, which are bacteria that have special nutritional requirement and usually proliferate only in a medium supplemented with mycobactin. The bacteria grow very slowly and require 7 to 11 weeks to form visible colonies.
In recent years, there has been suggested a method that enables early diagnosis of presence or absence of Mycobacterium avium subsp. paratuberculosis in feces by detecting a specific insertion sequence IS900 in DNA of Mycobacterium avium subsp. paratuberculosis by a polymerase chain reaction (PCR).
However, due to nonpersistent excretion of bacteria, an assured diagnosis was not completed because some individuals cannot be diagnosed or there may be “transited bacteria” which are excreted in feces without chance to invasion into intestinal tissue after oral intake of environmental bacteria in a contaminated farm. Therefore, there has been required an immunological diagnostic method to show evidence that a host is “really infected” with Mycobacterium avium subsp. paratuberculosis. 
As described above, paratuberculosis has a long period during which diagnosis is difficult, and all of conventionally prevalent methods of diagnosing paratuberculosis are lacking of assurance because the infection time or the like are limited due to its immunological properties. Therefore, there are still many paratuberculosis-infected animals, in particular, carrier animals, and the disease spread worldwide, which makes it difficult to eradicate paratuberculosis. 
Therefore, from the standpoint of animal hygiene, public hygiene, and livestock farming, there is an urgent need to realize prevention of paratuberculosis and eradication in the early stage of infection, and, in particular, there has been desired a diagnostic technique that enables diagnose of an infected animal in the immunological un-responsive stage difficult to diagnose the disease.
On the other hand, together with such direct economical damage due to paratuberculosis, it has recently been in the news that Mycobacterium avium subsp. paratuberculosis may be involved inhuman Crohn's disease (intractable disease designated by the Ministry of Health and Welfare of Japan) (Collins M T, Mycobacterium paratuberculosis: a potential food-borne pathogen? J Dairy Sci 80: 3445-8 (1997); Engstrand L, Mycobacterium paratuberculosis and Crohn's disease. Scand J Infect Dis Suppl 98: 27-9, 1995, and Momotani Eiichi “Relationship between Johne's disease and human Crohn's disease-Review-” Journal of Clinical Veterinary Medicine, vol. 19 (No. 7 (additional volume)), 2001).
Therefore, to clarify the relationship between paratuberculosis and human Crohn's disease, there has been required a diagnostic technique that enables diagnosis of infection of Mycobacterium avium subsp. paratuberculosis to human.
The present invention is intended to provide a diagnostic method for paratuberculosis by which an animal infected with Mycobacterium avium subsp. paratuberculosis can be sensitively diagnosed in the incubation period before the increase of specific antibody level and a large number of specimens can be treated.